两个油菜核因子Y的A亚基成员（NF-YA）参与油菜干旱调节的机制研究

Drought stress reduces rapeseeds yield by 15%~30% in canola (Brassica napus L).Two members of Nuclear Factor Y subunit alpha (NF-YAs) have been shown to play important role in canola drought response but its molecular mechanism was elusive according to our previous study. In the present project, differient length fragments from two NF-YA promoters were amplified and fused with Green Fluorescent Protein (GFP) or β-Glucuronidase (GUS) and further transformed to onion epidermal cells or canola to identify which fragments regulate two NF-YAs’ transcription. Besides, our experiments will explore if two BnNF-YAs are target of canola miRNA169 members by use of 5’ Rapid Amplification of cDNA Ends (5’RACE), tobacco transient expression and transgenic techniques. The direct downstream targets of two BnNF-YA members underlying drought stress would be elucidated through Chromatin Immunoprecipitation (ChIP) and Electrophoretic Mobility Shift assay (EMSA). The individual interacted proteins of two BnNF-YAs would be isolated and confirmed through Yeast Two-Hybrid (Y2H), Co-Immunoprecipitation (Co-IP) and Bimolecular Fluorescence Complementation (BiFC) and their roles in drought stress would be further analyzed. Accomplishing this study, we will characterize the molecular mechanism of two BnNF-YAs under drought stress and provide a clue for canola breeding.

干旱对油菜造成15%~30%的产量损失。申请者前期的研究发现两个油菜核因子Y的A亚基成员（BnNF-YA）参与植物的干旱调节，但对各自的分子机理均不清楚。本项目通过扩增两个BnNF-YA不同长度的启动子片段，分别和绿色荧光蛋白和β-葡萄糖苷酸酶报告基因连接，各自转入洋葱表皮细胞和油菜中表达，明确调节这两个油菜BnNF-YA的转录的启动子片段。通过5’cDNA末端快速扩增技术、烟草瞬时共表达和转基因技术明确这两个基因是否受油菜miRNA169家族成员的转录后调节。利用染色质免疫共沉淀和体外凝胶迁移技术来筛选两个油菜BnNF-YA的直接调节的下游基因。利用酵母双杂交、蛋白质免疫共沉淀和双分子互补实验筛选两个BnNF-YA的各自互作蛋白，并探索互作蛋白在植物耐旱中的作用。通过本项目的实施，可以明确油菜这两个BnNF-YA参与干旱调节的机理，从而为耐旱油菜的培育奠定理论基础。

非生物胁迫可以导致油菜的减产。本项目通过研究油菜中的2个NF-YA成员（NF-YA3和NF-YA9），发现这个两个基因可以调节油菜的逆境胁迫的能力。通过5’cDNA末端快速扩增技术、烟草瞬时共表达和转基因技术明确这两个基因受油菜miRNA169家族成员的转录后调节。通过克隆BnNF-YA不同长度的启动子片段，和β-葡萄糖苷酸酶报告基因连接，转入烟草叶片中表达，发现油菜BnNF-YA的转录不受启动子调控。利用酵母双杂交和免疫共沉淀实验等筛选了BnNF-YA的互作蛋白，筛选出的互作蛋白目前正在进行进一步的研究。本项目的研究为进一步研究这2个基因的功能打下良好的基础。